Generally in the biomedical arts, angiogenesis refers to the process of formation of new blood vessels from pre-existing ones (e.g., an existing monolayer of endothelial cells sprouting to form capillaries), whereas vasculogenesis is the term used for the formation of new blood vessels when there are no pre-existing ones (e.g., by a de novo production of endothelial cells). Angiogenesis plays a critical role in biologic processes such as organ development, wound healing, and tumor growth. It requires a well-orchestrated integration of soluble and matrix factors and timely recognition of such signals to regulate this process. Vasculogenesis plays a significant role during embryologic development, but can also occur in the adult organism. Circulating endothelial progenitor cells (derivatives of stem cells) can contribute to varying degrees to neo-vascularization during the early stages of tumor growth or in revascularization healing processes process following trauma (e.g., after cardiac ischemia). Thus, angiogenesis and vasculogenesis are critical for several normal and healthy physiological processes including embryogenesis, organogenesis and vascular remodeling. For ease of reference herein, both angiogenesis and vasculogenesis will collectively be referred to hereinafter as angiogenesis, and any reference to angiongenesis or use of words derived therefrom (e.g., “angiogenic,” “angiostatic,” etc.) should be interpreted in this collective fashion unless stated otherwise.
Angiogenesis, however, also is a fundamental step in the transition of tumors from a dormant state to a malignant one. Tumors are believed to induce blood vessel angiogenesis by secreting various growth factors (e.g., VEGF or bFGF), and/or by reducing the production of anti-growth factor enzymes (e.g., PKG). Such growth factors can induce capillary growth into the tumor, which research suggests helps supply the required nutrients that allow for tumor expansion and/or transports away waste products formed by rapidly dividing tumor cells. Angiogenesis thus is believed to be a key step for the transition from a small and harmless cluster of abnormal cells to a large tumor. Angiogenesis is also linked with the spread of a tumor, or metastasis. Single cancer cells can break away from an established solid tumor, enter the blood vessel, and be carried to a distant site, where those cells can implant and begin the growth of a secondary tumor. The subsequent growth of such metastases into secondary tumors will likewise require a supply of nutrients and oxygen and a waste disposal pathway—again implicating angiogenesis. Research has thus proceeded into the use of angiogenesis inhibitors in cancer treatments.
The angiogenesis process is regulated by a complex and interrelated system of pathways that involve various angiogenic and angiostatic factors. An imbalance of the angiogenic process can result in over or under expression of angiogenic or angiostatic factors, resulting in tumor vascularization and growth, or untimely termination of the angiogenesis process, resulting in unhealed chronic wounds.
Chemokines are a family of small proteins secreted by cells, which generally function as chemo-attractants, such as to guide the migration of cells. Members of the chemokine family are divided into four groups depending on the spacing of their first two cysteine residues. For example, the commonly used nomenclature in the art for referring to chemokine peptides having two adjacent cysteines near its amino terminus (or β-chemokine) is as a “CC-family” chemokine. As such, the name CCL1 would be used to denote the ligand 1 of the CC-family of chemokines, and the name CCR1 would be used to denote the receptor for CCL1. Likewise, the CXC-family of chemokines are a-chemokines, having their two N-terminal cysteines being separated by one amino acid, represented conventionally in its name with an “X,” and the ligands and receptors are represented using a similar CXCL# and CXCR# nomenclature.
Recent evidence demonstrates that members of the CXC chemokine family can act as either angiogenic or angiostatic factors, depending upon the presence of the ELR (Glu-Leu-Arg) motif in their NH2 terminus (see Strieter, R M et al., 1995, “The functional role of the ELR motif in CXC chemokine-mediated angiogenesis,” J. Biol. Chem. 270: 27348-57). Among the small CXC-family of chemokines are CXCL10 (also known as Interferon gamma-induced protein 10 or simply IP-10), CXCL11 (also known as IP-9/ITAC), and CXCL9 (Mig), which all lack the canonical N-terminal ELR sequence (see Godessart, N, et al., 2001, “Chemokines in autoimmune disease,” Curr. Opin. Immunol. 6: 670-675). These secreted proteins bind in common to the ubiquitous CXCR3 chemokine receptor, which is a seven transmembrane G-protein receptor that exists as two isoforms (CXCR3-A and CXCR3-B), which isoforms regulate chemotaxis and proliferation in a various cells types and acts as an angiostatic agent in endothelial cells (see Kelsen, S G et al., 2004, “The chemokine receptor CXCR3 and its splice variant are expressed in human airway epithelial cells,” Am. J. Physiol. Lung Cell Mol. Physiol. 287: L584-591). The A-isoform pf CXCLR3 has been found to be stimulatory, inducing both migration and proliferation, while the B-isoform inhibits migration and proliferation (see Lasagni, L. et al., 2003, “An alternatively spliced variant of CXCR3 mediates the inhibition of endothelial cell growth induced by IP-10, Mig, and I-TAC, and acts as functional receptor for platelet factor 4,” J. Exp. Med. 197:1537-1549; Bodnar, R J et al., 2006, “IP-10 blocks vascular endothelial growth factor-induced endothelial cell motility and tube formation via inhibition of calpain,” Circ. Res. 98:617-625; and Bodnar et al., 2009, “IP-10 induces dissociation of newly formed blood vessels,” J. Cell. Sci. 122:2064-2077). It has been proposed that CXCR3-A promotes chemotaxis and cell proliferation and CXCR3-B stimulates signals for growth inhibition (see Aidoudi, S et al., 2010, “Interaction of PF4 (CXCL4) with the vasculature: A role in atherosclerosis and angiogenesis,” Thromb. Haemost. 104: 941-948). Recent reports suggest that CXCR3 signaling results in chemotactic activation of keratinocytes via a PLCβ pathway that induces μ-calpain activation, which is mediated by calcium influx. In endothelial cells, however, chemotaxis is blocked via the inhibition of μ-calpain by a cAMP-PKA mediated pathway (see Bodnar, 2006, supra). Therefore, it is suggested that the regulation of these very different cellular responses is due to CXCR3-A/B binding of chemokines.
Furthermore, among the CXC-family of chemokines, CXCL10 specifically has been reported to be angiostatic and have antitumor activity via its signaling through CXCR3; resulting in inhibition of angiogenesis induced by vascular endothelial growth factor (“VEGF”) and basic fibroblast growth factor (“bFGF”), and in eventual in vitro and in vivo regression of nascent vessels (see Addison, C L et al., 2009, “The CXC chemokine receptor 2, CXCR2, is the putative receptor for ELR+ CXC chemokine-induced angiogenic activity,” J. Immunol. 165: 5269-5277; Bodnar, 2006, supra; and Bodnar, 2009, supra). In particular, newly forming vessels express CXC receptor 3 (“CXCR3”), and that activation of CXCR3 by its ligand CXCL10 both inhibits development of new vasculature and causes regression of newly formed vessels. CXCL10 is atypical, however, in that it specifically activates a single receptor (CXCR3) yet in several cells types induces motility while in others inhibit it (see Satish, L et al., 2005, “Interferon-inducible protein 9 (CXCL11)-induced cell motility in keratinocytes requires calcium flux-dependent activation of μ-calpain,” Mol. Cell. Biol. 5:1922-1941; Yates, C C et al., 2007, “Delayed and deficient dermal maturation in mice lacking the CXCR3 ELR-negative CXC chemokine receptor,” Am. J. Pathol. 1701: 484-495). For example, it has been reported that the CXCR3-binding chemokine CXCL10 can limit new vessel growth by inhibiting endothelial cell migration (see Bodnar, 2006, supra). In contrast, CXCL10 does not block the motility of keratinocytes but rather it increases their motility (see Yates, 2007, supra). Evidence suggests that this modulation occurs via the activation of two separate downstream pathways of CXCR3 (see Yates, C C et al., 2009, “Delayed reepithelialization and basement membrane regeneration after wounding in mice lacking CXCR3,” Wound Repair Regen. 17:34-41; Yates, C C et al., 2008, “ELR-negative CXC chemokine CXCL11 (IP-9/I-TAC) facilitates dermal and epidermal maturation during wound repair,” Am. J. Pathol. 173: 643-652; and Bodnar, 2006, supra]. CXCR3 thus is a part of a family of chemokine receptors that have opposing effects, and targeting one over the other can lead to regulation of specific cells or even cellular function in whole. There is an imperfect understanding in the art regarding exactly how and why CXCL10 and peptides derived therefrom may be used as a therapeutic, because the CXCL10 ligand binding to a single receptor can induce different biological effects.
CXCL10 is secreted by a diverse spectrum of tissues displaying pleiotrophic effects in immunity, angiogenesis, and organ-specific metastases of cancer, making it a promising therapeutic target for a wide variety of diseases. CXCL10 and CXCL11 also are known to be present in dermal wounds during the late transition from the regenerative to the resolving phase of wound healing. Specifically, CXCL11/IP-9 is expressed from re-differentiating basal keratinocytes behind the leading edge of a wound (see Satish et al., 2003, “ELR-negative CXC chemokine IP-9 as a mediator of epidermal-dermal communication during wound repair,” Journal of Investigative Dermatology, 120, 1110-1117), and CXCL-10/IP-10 is produced in the late healing state dermis where it is produced by endothelial cells (see Luster, et al., 1995, “The IP-10 chemokine binds to a specific cell surface heparan sulfate site shared with platelet factor 4 and inhibits endothelial cell proliferation.” Journal of Experimental Medicine, 182(1), 219-231). The timing and expression of IP-9 and IP-10, along with their cellular affects, has been determined by Applicants to provide a key communication between the dermis and epidermis during wound repair that, at least in part, signals an end to the regenerative phase and initiation of the remodeling phase of wound repair.
It has previously been determined that CXCL10 consists of three anti-parallel sheets overlayed by a helix at the C-terminus (see Swaminathan, G J et al., 2003, “Crystal structures of oligomeric forms of the IP-10/CXCL10 chemokine,” Structure 11: 521-532). It has been suggested that the N-loop region of the sheets play a role in binding of the protein to the receptor (see Clark-Lewis, I et al., 2003, “Structure-function relationship between the human chemokine receptor CXCR3 and its ligands,” J. Biol. Chem. 278:289-95), but the domain responsible receptor-activation has not been previously characterized such that it is fully understood.
To develop CXCL10 as a therapeutic agent, and, in particular, new and superior therapeutic peptides derived from CXCL10, structural details of its mechanism of action are needed to understand its role in the aforementioned pathological conditions. Further understanding of CXCL10 would potentially permit new peptides to be derived from the functional domain of CXCL10 that is responsible for CXCR3-B activation and related inhibition of endothelial cell function.
Thus, there remains a need in the art for improved therapeutics for the inhibition of angiogenesis that are highly effective in promoting cancer remission, preventing tumor malignancy and metastases, and in preventing progression of the disease to more advanced and aggressive stages.